The overall goal of this project is to apply new protein labeling strategies to better understand the dynamics of protein-DNA interactions. Protein-DNA interactions play important roles in many biological pathways, such as DNA transcription, translation, replication, recombination, and repair. To achieve specific protein-DNA interactions, a protein needs to search through megabases of non-target DNA. Proteins can search for specific targets by a 1D `sliding' motion, during which the protein remains in contact with the DNA, or via short-range diffusive `hopping' motions through 3D space. However, current imaging techniques focus on the 1-D translocation of the protein, while techniques for 3D mapping of protein translocation tracking rotational movements on DNA are scarce. In nanoplasmonic upconverting nanoparticles (UCNPs), the collective oscillation of conduction electrons, known as plasmons, lead to strong light absorption, as well as local field enhancements, which is dependent on the orientation of the nanoparticle with respect to the incident excitation. This upconversion fluorescence anisotropy of the nanoplasmonic UCNPs renders them as excellent orientation probes in both 2D and 3D. The UCNP labels could allow the continuous tracking of single-molecules in a variety of settings, which would increase our understanding of cellular function at the molecular level. To further advance our understanding of the biophysical mechanism underlying protein-DNA interactions, this project will use single particle orientation and rotation tracking of nanoplasmonic UCNP-labeled proteins to directly observe the curvilinear movement of proteins involved in the telomere maintenance and cohesion pathway. Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomere dysfunction has been strongly associated with degenerative diseases and cancer. Recent results from biochemical, cell based and single-molecule assays indicate that interactions of telomere maintenance proteins (including TRF1, TRF2) with telomeric DNA are dynamic. Recent studies show that cohesin subunit SA1 and TRF1 together function in promoting sister telomere association. Our preliminary results indicate that TRF1 and TRF2 carry out 1D diffusion, and suggest that SA1 hops on DNA. To advance our understanding of the biophysical mechanism underlying telomere maintenance, two proposed specific aims are: 1) To define the temporal and spatial resolution of movements of nanoplasmonic UCNP-labeled DNA-binding proteins using fluorescence microscopy. The shape anisotropy of the nanoplasmonic UCNPs creates changes in fluorescence intensity in the event of curvilinear motion. 2) Direct differentiation of curvilinear movement of TRF1/TRF2 and hopping of SA1 on DNA. We will compare single particle orientation and rotation tracking of nanoplasmonic UCNP- labeled TRF1 and SA1 proteins on DNA with random sequences and with telomeric DNA, with and without DNA lesions.